Gamma Interferon-inducible Lysosomal Thiolreductase (GILT) is the only known enzyme in the endocytic pathway that can reduce disulfide bonds in proteins. It is constitutively highly expressed in antigen presenting cells (APCs) and plays a key role in the processing of antigens containing disulfide bonds for presentation by MHC class II molecules to CD4+ T cells and, in dendritic cells, cross-presentation of antigens by MHC class I molecules to CD8+ T cells. GILT is also inducible by gamma interferon (IFN-?) and other inflammatory cytokines in most cell types. Homologues of GILT are present in many species that lack an adaptive immune system, suggesting that it may have functions in addition to its role on antigen processing. This proposal is based on our discovery of one such function: GILT is required for the optimal generation of the respiratory burst by macrophages exposed to bacterial lipopolysaccharide (LPS) or other pathogen-associated molecular patterns (PAMPs). Macrophages lacking enzymatically active GILT produce less reactive oxygen species (ROS) and are impaired in their ability to kill phagocytosed bacteria, including Salmonella typhimurium, Escherichia coli and Staphylococcus aureus. Mice in which the GILT gene is deleted are impaired in their ability to clear a peritoneal infection by Salmonella typhimurium. We propose to determine the mechanistic underpinnings of these phenomena. We will determine whether GILT influences the overall redox potential of the macrophage cytosol. We will determine if GILT expression influences the steady state intracellular distribution of the components of the dominant system responsible for ROS generation in macrophages and neutrophils, NADPH oxidase- 2 (Nox2), and whether it affects the changes in distribution of these components observed upon LPS stimulation and phagocytosis, with and without IFN-? stimulation. We will also determine the molecules in macrophage lysosomes and phagosomes that are targets for GILT activity, with a particular focus on Nox2 components that are luminally exposed. We will also determine the potential role of GILT in ROS generation by other cell types. These include the prototypical cell type that uses Nox2, the neutrophil, and other cell types that can generate ROS using homologues of Nox2. Some of these homologues share regulatory components with Nox2 but others do not. These studies will allow us to determine if GILT plays a general role in ROS generation, and to determine the molecular mechanism by which it acts.